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This information is current as of June 18, 2017. Regulation of T Lymphocyte Trafficking into Lymph Nodes During an Immune Response by the Chemokines Macrophage Inflammatory Protein (MIP)-1 α and MIP-1β Nicodemus Tedla, Hong-Wei Wang, H. Patrick McNeil, Nick Di Girolamo, Taline Hampartzoumian, Denis Wakefield and Andrew Lloyd J Immunol 1998; 161:5663-5672; ; http://www.jimmunol.org/content/161/10/5663 Subscription Permissions Email Alerts This article cites 50 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/161/10/5663.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References Regulation of T Lymphocyte Trafficking into Lymph Nodes During an Immune Response by the Chemokines Macrophage Inflammatory Protein (MIP)-1a and MIP-1b1 Nicodemus Tedla, Hong-Wei Wang, H. Patrick McNeil, Nick Di Girolamo, Taline Hampartzoumian, Denis Wakefield, and Andrew Lloyd2 T he migratory properties of leukocytes have evolved to allow efficient surveillance of tissues for infectious pathogens and rapid accumulation at sites of injury or infection. In contrast to neutrophils and monocytes, T lymphocytes may exit from the vascular compartment via specialized high endothelial venules (HEV)3 in lymphoid organs and recirculate many thousands of times during their life span (1, 2). It has been estimated that one in every four lymphocytes leaves the circulation by crossing the HEV (3). After encountering Ag in lymph nodes, memory T lymphocytes continually patrol the body for that Ag by recirculating from the blood, through tissues, into the lymphatic system, and back to the blood (1– 4). T lymphocytes thus acquire a predilection, based on the environment in which they first encounter Ag, to home to, or recirculate through, that same environment (5, 6). Hence, relatively distinct subsets of T lymphocytes extravasate through the microvasculature in tissues such as skin and gut and across HEV in lymph nodes (4 – 6). The arrival of Ag, and hence the induction of an immune response in the node, greatly increases blood flow and traffic of lymphocytes across HEV, coupled with a transient, sharp decrease in recirculating lymphocyte output from the efferent lymphatics (7– 9). In lymph nodes undergoing an immune response, lymphocyte Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, Australia Received for publication April 14, 1998. Accepted for publication July 13, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work is supported by the National Health and Medical Research Council of Australia. 2 Address correspondence and reprint requests to Dr. Andrew Lloyd, Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, NSW, 2052, Australia. 3 Abbreviations used in this paper: HEV, high endothelial venules; VLA, very late Ag; DNFB, 2,4-dinitrofluorobenzene; MIP, macrophage inflammatory protein; MCP, monocyte chemotactic protein; MMCP-5, mouse mast cell protease-5; PCNA, proliferating cell nuclear Ag; TBS, Tris-buffered saline; R-PE, R-phycoerythrin. Copyright © 1998 by The American Association of Immunologists traffic across the HEV may increase substantially within 3 h after antigenic stimulation and by as much as 10-fold over the first 48 h of the response (8, 9). This effect is not caused by lymphocyte proliferation within the node nor by increased numbers of cells entering the lymph nodes from the lymphatics; instead, .95% of the effect is due to trafficking of cells from blood (8, 9). At the molecular level, the leukocyte adhesion molecules, Lselectin, LFA-1 (CD11a/CD18), and VLA-4 (CD49d/CD29), mediate T lymphocyte binding to peripheral lymph node HEVs by interaction with glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) (10) or CD34 (11) for L-selectin, with ICAM-1 and ICAM-2 for LFA-1 (12, 13), and potentially with fibronectin for VLA-4 (14). Neutralizing antisera to L-selectin (15, 16), LFA-1 (17), or VLA-4 (14) markedly reduce lymphocyte migration into peripheral lymph nodes. Thus, lymphocyte recruitment into lymph nodes is likely to be a multistep process (similar to the processes of neutrophil and monocyte localization in inflammation) that requires L-selectin molecules to allow lymphocytes to “tether and roll’’ via low affinity interactions and LFA-1 or VLA-4 to induce firm adhesion to their counterreceptors (4, 18, 19). Activation of L-selectin alone has been shown to trigger the high affinity state of integrins on naive T lymphocytes in vitro (20, 21), thus providing a potential mechanism for the preferential recirculation of these cells through peripheral lymph nodes. However, pertussis toxin treatment abrogates LFA-1-dependent arrest of lymph node-derived lymphocytes (22) and may inhibit lymphocyte entry into secondary lymphoid organs (23), suggesting a requirement for G protein-linked signaling events in T lymphocyte trafficking into lymph nodes. Lymphocyte chemoattractants secreted from within peripheral lymph nodes and their G protein-coupled receptors expressed on lymphocyte subpopulations may provide this signal to stimulate integrin-dependent recruitment of lymphocyte subsets. Members of the b-chemokine family are known to direct T lymphocyte migration along a protein gradient (chemotaxis) (24, 25) and to induce adhesion to extracellular matrix proteins (26). The b-chemokines MIP-1a, MIP-1b, monocyte chemotactic protein 0022-1767/98/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation. Their role in regulation of T lymphocyte traffic into lymph nodes during the development of an immune response has not previously been explored. The sensitization phase of contact hypersensitivity induced by the hapten, dinitrofluorobenzene (DNFB) in the mouse was used as a model of T lymphocyte trafficking in response to antigenic stimulation. Rapid accumulation of CD81 and CD41 T cells in the draining lymph nodes was closely associated with strongly enhanced expression of macrophage inflammatory protein (MIP)-1a and MIP-1b mRNAs and proteins. Mast cells accumulating in the nodes during DNFB sensitization were the predominant source of MIP-1b, whereas MIP-1a was expressed by multiple cell types. Neutralization of these chemokines profoundly inhibited T lymphocyte trafficking into lymph nodes and altered the outcome of a subsequent challenge to DNFB. Thus, b-chemokines regulate T lymphocyte emigration from the circulation into lymph nodes during an immune response and contribute significantly to the immunologic outcome. The Journal of Immunology, 1998, 161: 5663–5672. 5664 CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES Lymph node excision Both right and left inguinal lymph nodes were removed by carefully dissecting the tissue to a radius of approximately 1 cm from the center of the node. The right inguinal lymph node of each animal was used to prepare a single-cell suspension, and the left inguinal lymph node was fixed in 10% buffered formalin (pH 7.0) and embedded in paraffin. Additional mice were used to harvest the right inguinal node for extraction of protein from tissue lysates, and the left inguinal node was snap frozen by embedding the tissue in Tissue-Tek (OCT compound, Miles, Elkhart, IN) before storage at 270°C for subsequent immunohistochemical studies. Preparation of the single-cell suspension Materials and Methods A cell suspension from each right inguinal lymph node was prepared using a modification of a previously published method (34). In brief, the lymph node was weighed and washed twice in RPMI 1640/10% FCS supplemented with penicillin/streptomycin and L-glutamine. The tissue was minced to nearly complete dissociation using scissors and forceps in a 6-well cell culture dish (Costar, Cambridge, MA) and resuspended in 4 ml of the above-mentioned medium. One mg/ml of collagenase type H (Boehringer Mannheim, Berlin, Germany) containing 1 mM calcium chloride was added to the mince, which was then incubated at 37°C for 60 min. Digestion was then stopped by adding 1 ml of RPMI 1640/20% FCS and the suspension filtered through a 40-mm nylon cell strainer (Becton Dickinson, Mountain View, CA) into a 50-ml Falcon tube. A tuberculin syringe plunger was used to tease cells from tissue on top of the nylon mesh with repeated rinsing in PBS. The cells were washed twice with PBS and resuspended in this medium at 2 3 106/ml. The total cell count was enumerated after assessment of viability with trypan blue. Mice Measurement of differential leukocyte counts in the lymph nodes Six- to eight-week-old male and female C3H/HeN mice were bred in specific pathogen free (SPF) conditions in the Animal Breeding and Holding Unit at the University of New South Wales. SPF conditions were maintained throughout the experimental phase of the study. For the experiments described, each sampling point consisted of four mice. From each single-cell suspension, four air-dried smears were made. One slide from each mouse was then stained with Giemsa-Grunwald to determine a differential leukocyte count. The remaining three slides were fixed in acetone and stored at 220°C for immunohistochemical staining. Sensitization of contact hypersensitivity Twenty-five microliters of 0.5% 2,4-DNFB (Sigma, Sydney, Australia) in 4/1 diluted acetone/olive oil (Sigma) was applied to the freshly shaved abdominal surface of mice on day 0 and day 1 for sensitization (33). Control mice were painted on the shaved abdomen with 25 ml of the vehicle alone. Elicitation of contact hypersensitivity and evaluation of ear swelling Five days after sensitization, mice were challenged by applying 10 ml of 0.2% DNFB in acetone/olive oil to one ear. Control mice were similarly painted with the vehicle alone. The degree of ear swelling was measured before challenge and 24 h postchallenge using an engineer’s micrometer (Mitutoyo, Tokyo, Japan). Each earlobe was measured twice and contact hypersensitivity determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear, expressed in micrometers (mean 6 SD). Mice that were challenged with the hapten without previous sensitization served as an additional negative control. Antichemokine treatment in vivo Four hours before DNFB sensitization, neutralizing goat anti-mouse polyclonal Abs directed against murine MIP-1a and/or MIP-1b were injected i.p., diluted in sterile endotoxin-free saline, and control animals were injected with control goat IgG (R&D Systems, Minneapolis, MN) in comparable doses to the experimental animals. Mice were coded so that cell counts obtained during the sensitization phase and ear thickness measurements after challenge were made by an independent observer without knowledge of the status of the mouse. Peripheral blood collection Anticoagulated blood (200 –500 ml) was collected from the heart of each mouse 1–2 min after the animals were sacrificed by CO2 inhalation, and a vertical neck-to-tail skin incision was performed. Two sets of thin blood films were made for differential counting after Giemsa-Grunwald staining, and the total cell count was measured by automated cell counter (Sysmex NE 800, Australian Diagnostic Services, Sydney, Australia). Blood (50 ml) was used to stain cell surface markers with mAbs in three-color flow cytometry (see below). Three-color flow cytometry The Abs use in flow cytometry included: anti-CD3-FITC, anti-CD4-Rphycoerythrin (R-PE), and anti-CD8a-RED163, which were rat anti-mouse mAbs purchased from Life Technologies (Victoria, Australia). An irrelevant negative control IgG with subclasses g1 and g2a (IgG1-FITC/IgG2aPE) and the FACS lysing solution were purchased from Becton Dickinson. The wash solution consisted of PBS containing 2% BSA and 0.2% sodium azide. A 1% paraformaldehyde solution in PBS was used to fix cells after staining. mAb (4 ml) and ;2 3 105 cells in 100 ml were added to each labeling tube. After mixing, tubes were incubated in the dark at 4°C for at least 30 min, then FACS lysing solution was added and tubes were further incubated at room temperature for 10 min followed by centrifugation. The supernatant was decanted and the cell pellet washed and then fixed in paraformaldehyde solution. A total of 10,000 events were acquired using a FACScan flow cytometer and data analysis performed with PC LYSIS II software (Becton Dickinson). Analysis of proliferating lymphocytes in vivo To determine the proportion of proliferating lymphocytes in response to DNFB sensitization, as opposed to the cells recruited into the lymph nodes, a mAb against proliferating cell nuclear Ag (PCNA) conjugated to FITC was purchased from Boehringer Mannheim (Mannheim, Germany). Lymph node-derived single-cell suspensions (106/ml) were fixed for 2 min in 1% paraformaldehyde, followed by washing in cold PBS. Cells were then incubated in 100% methanol at 220°C for 10 min, centrifuged again, and washed in PBS containing 0.1% Triton X100 (Serva, Heidelberg, Germany). Subsequently, cells were incubated with anti-PCNA Ab (1.25 mg in 50 ml of 2% BSA in PBS) for 15 min at room temperature. After washing in PBS supplemented with 2% BSA, the cells were spun down and incubated with anti-CD4-R-PE and anti-CD8a-RED163 Abs for at least 30 min at 4°C in the dark. Cells were then washed twice in PBS/2% BSA and resuspended in PBS. As a positive control for this analysis, lymph nodederived mononuclear cells were stimulated in vitro with 10 mg/ml of phytohemagglutinin (Wellcome Diagnostics, Charlotte, NC) before staining as described above. Samples were analyzed on a FACScan (Becton Dickinson) equipped with LYSIS II software. In situ hybridisation Single-stranded cRNA probes of 350 bp in both the antisense and sense orientation were prepared by in vitro transcription from murine MIP-1a Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 (MCP)-1, MCP-2, MCP-3, and RANTES reportedly show chemotactic activity for T cells in vitro (27). Of these chemokines, some in vitro studies suggest that MIP-1a and MIP-1b preferentially attract CD81 and CD41 T cells, respectively (24, 25, 28). We have recently reported the first evidence for a potential role of chemokines in the regulation of lymphocyte traffic into lymph nodes in studies of nodes taken from HIV-1-infected patients and control subjects (29). Strong expression of MIP-1a in the HIV lymph nodes was associated with the accumulation of CD81 T cells in these tissues. The present experiments were designed to further define the potential in vivo role of MIP-1a and MIP-1b in recruitment of T cell subsets to lymph nodes during an immune response. The well-characterized murine model of contact hypersensitivity induced by dinitrofluorobenzene (DNFB) was chosen for this study. It is believed that both CD41 and CD81 T cell subsets are involved in the development of contact hypersensitivity mediated by DNFB, because studies to define the specific role of either subset have provided conflicting results (30, 31, 32). The pattern of expression of MIP-1a and MIP-1b mRNAs and proteins in draining lymph nodes of DNFB-painted mice was examined, and the kinetics of accumulation of T lymphocyte subsets in the nodes was determined. The Journal of Immunology 5665 and MIP-1b plasmid cDNAs using a nonisotopic probe-labeling technique (digoxigenin; Boehringer Mannheim). After a 2-h prehybridization at 42°C, hybridization was performed overnight at the same temperature using 100 ng of probe in 25 ml of prehybridization solution on 4-mm thick, formalin-fixed, paraffin-embedded sections. This was followed by repeated stringency washing at 42°C in 0.53 SSC. These hybridization and washing conditions were empirically determined to give optimal signal with the antisense probes, but minimal nonspecific signal with the control (sense strand) probes. Probe detection was conducted according to the manufacturer’s directions with an anti-digoxigenin mAb conjugated to alkaline phosphatase followed by an appropriate substrate (nitro blue tetrazolium 1 59-bromo-4-chloro-3-indolyl phosphate (NBT 1 BCIP); Boehringer Mannheim). Control samples included sections of lymph nodes of the DNFB-treated mice hybridized without probe or without Ab detection, as well as sections obtained from the lymph nodes of acetone-treated mice. Immunohistochemical staining Quantitative evaluation of MIP-1a and MIP-1b mRNA expression After a systemic sampling procedure as described elsewhere (29), computer-assisted morphometric analysis of lymph node sections was used to quantitate the number of cells expressing chemokine mRNAs as well as the total number of mast cells in the nodes (36). In brief, contiguous fields across the whole section (on average, 10 fields) at a final magnification of 2503 were assessed. After ensuring that the sections hybridized with the sense probes exhibited no significant signal, the number of positive cells (cytoplasmic blue staining) per field was enumerated. Although significant regional variations in staining were observed, the mean count for the whole section is reported as a conservative measure of the signal for each probe. Morphologic analyses and immunohistochemical staining of adjacent sections were used to determine the cellular sources of MIP-1a and MIP-1b. Analysis of MIP-1a and MIP-1b production by Western blotting Cell lysates from groups of three mice sacrificed 4, 24, and 96 h after DNFB repainting, and 24 h after acetone/olive oil repainting (control mice), were prepared using a standard method. To ensure equal loading of protein, the amount of total protein in the cell lysate was measured using the bicinchonic acid protein assay kit (Pierce, Rockford, IL). Lymph node-derived cell lysates were electrophoretically separated on a 4% stacking and 10% resolving acrylamide gel under nonreducing conditions and then transferred to Immobilon-P membranes (Millipore, Sydney, Australia) using a Trans-blot SemiDry Electrophoretic Transfer Cell (Bio-Rad, Sydney, Australia). After protein transfer, the membranes were washed twice in Tris-buffered saline (TBS) for 15 min at room temperature. Membranes were then incubated overnight at 4°C with 50 ml of blocking solution containing 3% BSA and 5% skim milk powder in TBS. The membranes were then rinsed twice with TBS for 5 min at room temperature and incubated with the primary goat anti-mouse MIP-1a or MIP-1b Ab (R&D Systems) for 1 h at room temperature with continuous gentle shaking. Primary Abs were reconstituted in sterile PBS at a concentration of 1 mg/ml and used at a 1:500 dilution. After incubation with the primary Ab, the membrane was washed four times for 15 min with TBS and incubated for 1 h at room temperature with a 1:500 dilution of donkey anti-goat second- FIGURE 1. DFNB sensitization produces a rapid increase in the weight and cellularity of inguinal lymph node (A) and a decrease in the peripheral blood leukocyte count (B). The right inguinal lymph node collected from animals sensitized by painting with 0.5% DNFB and control animals treated with vehicle alone (acetone) were weighed, minced, and digested with collegenase. Single-cell suspensions prepared from each node were counted both manually and with an automated cell counter (Sysmex NE 800). Cell smears prepared from the single-cell suspension were used for differential cell counting after Giemsa-Grunwald staining. A total leukocyte count was also performed, using the automated cell counter, in peripheral blood collected from each animal. Each experimental group consisted of four animals, and each experiment was performed twice. “Acetone” on the x-axis represents pooled data from two sets of four control animals that were sacrificed 4 and 24 h after repainting with vehicle alone. Results are presented as mean 6 SD. ary Ab (Serotec), which was directly conjugated to horseradish peroxidase. Membranes were then washed four times for 15 min in TBS, followed by the addition of a chemiluminescence reagent (Renaissance, DuPont, Sydney, Australia). The membranes were finally exposed to X-OMAT-AR scientific imaging film (Kodak, Sydney, Australia). Metachromatic staining After dewaxing and a 10-min incubation with 0.5 N hydrochloric acid, overnight staining with 1% toluidine blue in 0.5 N HCL was used as a metachromatic stain for mouse mast cells in the formalin-fixed lymph node tissue sections. A standard hematoxylin and eosin staining method was used for evaluation of the histopathologic changes in the lymph node. Statistical analysis A computer software program, Microsoft EXCEL, version 5.0 (Microsoft, Seattle, WA), was used to calculate means and SD. To assess the level of statistical significance, a two-tailed Mann-Whitney U test was performed using a statistical software program, SPSS for Windows, version 6.0 (SPSS, Chicago, IL). Parameters of interest in each group of DNFB-treated Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 A standard two-step streptavidin-horseradish peroxidase staining technique was performed to identify cell types on frozen sections and smears using primary rat mAbs against mouse T cell Ags CD3, CD4, CD8; a B cell marker, CD40; and a marker for tissue macrophages (Mac-1); as well as an isotype-matched negative control Ab. A biotinylated rabbit anti-rat secondary Ab was used. All of the above Abs were purchased from Serotec (Australian Laboratory Services, Sydney, Australia). A polyclonal rabbit anti-mouse Ab directed against mast cell protease-5 (MMCP-5; Ref. 35) was used to stain mast cells in formalin-fixed lymph node tissues after an enzymatic digestion and Ag retrieval by microwave treatment of the sections in 0.01 M sodium citrate buffer, pH 6.0. A biotinylated goat antirabbit secondary Ab was used for this staining (Dako, Glostrup, Denmark). Immunohistochemical staining using primary goat anti-mouse polyclonal Abs directed against MIP-1a and MIP-1b (R&D Systems) and secondary donkey anti-goat IgG directly conjugated to horseradish peroxidase (Serotec) were used to detect MIP-1a and MIP-1b proteins in formalin-fixed sections after a similar enzymatic digestion and Ag retrieval by microwave of the sections in citrate buffer. An isotype-matched goat IgG (R&D Systems) was used as a negative control. A standard two-step streptavidin-horseradish peroxidase staining technique was performed on formalin-fixed, paraffin-embedded lymph node sections to localize proliferating cells using primary anti-PCNA mAb (Dako) and a biotinylated goat anti-mouse secondary Ab (Serotec). 5666 CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES Table I. Induction of cell proliferation in draining lymph nodes of mice treated with DNFB a Treatment Mean Lymphocyte Count per Lymph Node (3106) CD41 CD81 Acetone 4 h after DNFB 24 h after DNFB 96 h after DNFB PHA-stimulated lymphocytes 1.5 6 0.2 3.4 6 0.5 7.9 6 1.0 3.2 6 0.5 — 1.5 6 0.4 2.4 6 0.5 2.4 6 0.2 14.3 6 2.7 99.2 6 5.8 1.8 6 0.6 2.7 6 0.8 2.2 6 0.7 10.1 6 0.4 98.4 6 5.5 % PCNA1b FIGURE 2. DFNB sensitization produces a rapid increase in CD41 and CD81 T lymphocytes in lymph nodes (A) and an associated decrease in the peripheral blood (B). Cells (2 3 105) of the single-cell suspension from the inguinal lymph node and heparinized whole blood obtained from each animal were stained with rat anti-mouse CD3-FITC, CD4-R-PE, and CD8RED613 mAbs. To obtain the proportions of the T cell subsets, three-color flow cytometric analysis was performed on the lymphocyte gate based on forward and side scatter as well as the T cell (CD3) gate. The absolute values for the T cell subsets were then obtained from the total leukocyte count, the proportions of total lymphocytes (obtained from the differential count), and the proportions of the T cell subsets (obtained from the flow cytometry). Isotype-matched irrelevant rat IgG Abs conjugated to the three different fluorochromes were used as negative controls. Each experimental group consisted of four animals, and each experiment was performed twice. “Acetone” on the x-axis represents pooled data from two sets of four control animals that were sacrificed 4 and 24 h after repainting with vehicle alone. Results are presented as mean 6 SD. animals were analyzed for statistical significance in comparison with control animals. As several comparisons were performed, Bonferoni adjustments for statistical significance were made. Results DNFB sensitization causes a rapid increase in cellularity and weight of inguinal lymph nodes The lymph node weight increased significantly within 30 min of repainting with DNFB ( p , 0.01), peaking at 12 h and gradually decreasing thereafter (Fig. 1A). The lymph node weight remained mildly increased above that of the control animals 1 mo after repainting. The enlargement of the lymph nodes was accompanied by a .10-fold increase in the number of nucleated cells in the nodes (Fig. 1A). Differential cell counting after Giemsa-Grunwald staining of the cell smears showed that .90% of the cells in lymph nodes of DNFB-painted mice were lymphocytes, confirming that these cells represented the predominant leukocyte subpopulation accumulating in the nodes. A substantial proportion of the increase in lymphocyte numbers was evident within 30 min, with ;85% of the peak increase detected within 12 h (Fig. 1A). This rapid accumulation was statistically significant ( p , 0.01) when compared with the control animals. The brisk kinetics indicate increased lymphocyte traffic into the nodes (and perhaps reduced output) rather than in situ proliferation of lymphocytes. DNFB repainting induces a rapid drop in PBL counts Further evidence in support of the notion of increased trafficking of lymphocytes into lymph nodes was obtained from the rapid decrease in lymphocyte numbers in peripheral blood coincident with their accumulation in the lymph nodes. Sensitization with DNFB produced an ;50% reduction in the total number of PBL within the first 30 min after repainting (Fig. 1B). The count gradually returned to normal values within 48 h after the treatment. CD41 and CD81 T lymphocytes accumulate in lymph nodes after repainting with DNFB Three-color flow cytometric analysis of the draining lymph nodes of DNFB-treated mice showed a significant increase in the total (CD31) T lymphocytes in lymph nodes, which peaked within 12 to 24 h after repainting (Fig. 2A). Both CD41 and CD81 T lymphocyte subsets showed a similar rapid increase ( p , 0.01 for each) following repainting with DNFB. After reaching a peak at 24 h, the CD41 and CD81 lymphocyte numbers in the nodes of DNFBtreated mice remained significantly higher than those of control lymph nodes 28 days after the repainting. The proportions of PCNA-positive CD41 and CD81 T cell subsets, however, remained ,2.5% (Table I) over the first 24 h, suggesting that the marked increase of both T cell subpopulations was predominantly attributable to recruitment rather than proliferation in situ. In the peripheral blood, there was a rapid and marked drop in the number of total T lymphocytes (CD31), .50% in the first 30 min, which gradually recovered within 24 to 48 h posttreatment (Fig. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 a Sensitization was performed on day 0 and 1 by painting hapten or acetone on the shaved abdomen as described elsewhere (see Materials and Methods). Lymphocytederived single-cell suspensions (1 3 106/ml) were fixed in 1% paraformaldehyde for 2 min. After washing in cold PBS, cells were incubated in 100% methanol at 220°C for 10 min and washed again with cold PBS containing 0.1% Triton X-100. Subsequently, cells were incubated with FITC-conjugated anti-PCNA Ab (1.25 mg in 50 ml of 2% BSA in PBS) for 15 min at room temperature. After washing in PBS supplemented with 2% BSA, cells were further incubated with anti-CD4-R-PE and antiCD8-RED613 Abs at 4°C for 30 min in the dark. Cells were then washed twice in PBS/2% BSA and resuspended in 0.5 ml of PBS. As a positive control for this analysis, lymph node-derived mononuclear cells (.70% T lymphocytes) were stimulated for 3 days in vitro with 10 mg/ml of PHA. Samples were analysed on a FACScan equipped with LYSIS II software. Each group consisted of four animlas. Results are expressed as mean 6 SD. b Represents the proportion of PCNA-positive cells (mean 6 SD) of the total CD41 or CD81 T cell populations. The Journal of Immunology 5667 2B). This reduction included both the CD41 and CD81 populations (Fig. 2B). the paracortex was also noted. Increased activity of germinal centers was visible in some nodes (data not shown). DNFB sensitization induces parafollicular hyperplasia and sinus histiocytosis of draining lymph nodes MIP-1a and MIP-1b are rapidly induced during DNFB sensitization During the period of lymph node enlargement after DNFB repainting (0 – 4 days), histologic examination revealed prominent subcapsular and medullary sinus expansion (data not shown). These regions were filled with a large number of leukocytes, predominantly lymphocytes, macrophages, and mast cells, but also polymorphonuclear cells. At later time points, there was also a significant enlargement of the hilar region and a slight expansion the medullary regions of the nodes. A less pronounced expansion of In situ hybridization and immunohistochemical staining of the draining lymph nodes demonstrated the induction of MIP-1a and MIP-1b mRNAs (Fig. 3) and abundant production of these proteins in animals sensitized with DNFB (Fig. 3). By contrast, lymph nodes obtained from acetone-painted (control) mice showed no expression of these chemokine mRNAs (Figs. 3 and 5). Computerassisted morphometric analysis of lymph node sections to quantitate the number of cells expressing chemokine mRNA, confirmed Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 3. DFNB sensitization rapidly induces expression of MIP-1a and MIP-1b mRNAs in draining lymph nodes. A and B, Sections (1603 magnification) from animals sacrificed 4 and 24 h after DNFB repainting, showing intense blue staining of MIP-1b mRNA-expressing cells located in the subcapsular and hilar regions. The background is lightly counterstained with neutral red. C, Lymph node section from a mouse sacrificed 4 h after repainting with DNFB, hybridized with the MIP-1a probe, showing abundant expression of the chemokine mRNA (blue staining) in the hilar and medullary regions. D, Section obtained 24 h after repainting with DNFB, showing substantial parafollicular staining of MIP-1a mRNA. Hybridization of adjacent sections to those shown in A–D with the sense MIP-1b amd MIP-1a probes showedno signal (data not shown). E, A lymph node section from an acetone-painted (control) mouse hybridized with the atisense MIP-1a probe showing no mRNA signal. No positive signal was found in control lymph node sections hybridized with the antisense MIP-1b probe (data not shown). F, Immunohistochemical stain (positive cells stained red) with anti-MIP-1a Ab on a section from an animal sacrificed 24 h after repainting with DNFB. 5668 CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES the maximal induction of both chemokines at 4 h (Fig. 4A). As expected, the kinetics of induction of the chemokine proteins in Western blot analyses was slightly delayed in comparison with the mRNA. MIP-1b expression was faintly evident in control tissues and was relatively constant from 4 –96 h after DNFB treatment (Fig. 4B, upper panel), whereas MIP-1a expression appeared maximal at 24 h (Fig. 4B, lower panel). Thus, the expression of these chemokine proteins was coincident with the accumulation of CD41 and CD81 T lymphocyte subsets, suggesting a functional role for these chemokines in the regulation of T lymphocyte recruitment. MIP-1b is expressed predominantly by mast cells, whereas MIP-1a is expressed by a wide range of lymph node cells Treatment with anti-chemokine Abs abrogates T cell recruitment to the draining lymph nodes To test whether T lymphocyte recruitment in this model was dependent on MIP-1a and MIP-1b, mice were given an i.p. injection of neutralizing anti-chemokine Abs 4 h before the first painting with DNFB (37, 38). The anti-chemokine Abs caused a dose-dependent inhibition of the recruitment of T lymphocytes to the draining lymph nodes. Administration of 50, 200, or 500 mg of anti-MIP-1a Ab reduced CD31 T cell numbers, evaluated at 24 h after repainting, by 16% ( p , 0.05), 31% ( p , 0.01), or 48% ( p , 0.01), respectively, of the cell numbers in animals injected with 1000 mg of control Ab. Similarly, anti-MIP-1b Ab treatment in the same doses produced 10, 20, and 49% inhibition. The inhibition produced by pretreatment of mice with 500 mg of either anti-MIP-1a or anti-MIP-1b Abs was significant for both CD41 and CD81 T cells ( p , 0.01 for all four comparisons; Fig. 6). Treatment with anti-chemokine Abs modifies a subsequent delayed-type hypersensitivity response To assess the effect of chemokine inhibition on the outcome of the DNFB-induced immune response, mice treated with a combination of anti-MIP-1a (500 mg) and anti-MIP-1b (500 mg) Abs before DNFB sensitization were challenged 5 days later with 0.2% DNFB applied to the left ear. Mice that were injected with control Ab exhibited a good ear swelling response, comparable with that of positive control animals, whereas mice pretreated with a combination of anti-MIP-1a and anti-MIP-1b Abs before hapten appli- FIGURE 4. Chemokine mRNA and protein expression in draining lymph nodes increases dramatically during DNFB sensitization. A, In situ hybridization was performed using MIP-1a or MIP-1b riboprobes. Contiguous high power fields across each whole section were examined before the mean number of cells per high power field (6SEM) was enumerated with a computer-assisted analysis system. Two lymph node sections from each mouse (four mice per time point) were analysed. B, Immunoblotting from chemokines in cell lysates prepared from mouse lymph nodes using anti-MIP-1b Ab (upper panel) demonstrated the production of an ~8.5-kDa MIP-1b protein at 4, 24, and 96 h after repainting with DNFB. The 8.3-kDa band of the standard m.w. marker (Bio-Rad) is indicated on the left side of the membrane. Immunoblotting of cell lysates derived from mouse lymph nodes using an anti-MIP-1a Ab (lower panel) demonstrated the production of an ~8.5-kDa protein at 4 h (lane 2) and 24 h (lane 3) but not at 96 h (lane 4) after repainting with DNFB. Lysates from acetone-painted (control) mice (lane 1) showed no detectable MIP-1a. Each lane was loaded with an equal amount (20 mg) of cell lysate derived from a pool of four to five animals. cation showed a modest, but significant, 19% inhibition of the contact hypersensitivity reaction elicited by ear lobe challenge (Table II). Discussion A considerable body of in vitro and in vivo data highlights the role of chemokines in the regulation of leukocyte emigration from the vascular compartment to sites of inflammation (27). This chemokine regulation hinges upon the ability of these cytokines to induce affinity modulation of integrin adhesion molecules expressed on leukocytes (4, 13, 27, 39) and upon the target cell specificity of the individual chemokines (27, 39 – 41). Like other leukocyte subpopulations, T lymphocytes appear to depend on chemokine-regulated mechanisms for recruitment across vascular endothelium to sites of inflammation (23, 27). The present findings provide clear in vivo evidence for an association between the production of the b-chemokines MIP-1a and MIP-1b and the recruitment of CD41 and CD81 T lymphocytes into peripheral lymph nodes during an immune response. In this model of contact hypersensitivity, T lymphocytes accumulated Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Histomorphologic and immunohistochemical studies of lymph node sections revealed the cellular sources of MIP-1b mRNA were significantly different from those of MIP-1a. At all time points, a range of cell types in the nodes was found to express MIP-1a, including macrophages, lymphocytes, and endothelial cells (figure not shown). By contrast, a striking and novel finding was the demonstration that mast cells were the predominant source of MIP-1b mRNA in the lymph nodes of DNFB-treated mice (Fig. 5). Furthermore, computer-assisted quantitation in the nodes confirmed a significant increase in the number of mast cells in the subcapsular and hilar regions of the draining nodes (54). This increase was concurrent with a decrease in the number of mast cells at the site of sensitization in the skin, thus suggesting that mast cells travel from the skin via afferent lymphatics. Immunohistochemical detection of MIP-1b protein confirmed the mast cell localization of this chemokine. Furthermore, prominent staining of MIP-1b was found on endothelial cells (Fig. 5D), despite the absence of detectable mRNA in these cells. As the accumulation of MIP-1bexpressing mast cells coincided with the accumulation of T lymphocytes in the nodes, this pathway may be critical to the observed rapid recruitment of T lymphocytes into the nodes after DNFB repainting. The Journal of Immunology 5669 very rapidly within the nodes and were predominantly PCNA negative, thereby precluding any significant role for lymphocyte proliferation as an explanation for the 10-fold increase in T lymphocyte numbers observed during sensitization. The notion of altered trafficking of leukocytes into lymph nodes during the induction of contact hypersensitivity was supported by the rapid decrease in T lymphocyte numbers in the peripheral blood coincident with their accumulation in the lymph nodes. In a similar fashion, a significant decrease in the PBL count coincident with accumulation of these cells in the lymph nodes was observed in mice that had received repeated i.p. injections of Corynebacterium granulosum (42). Three-color flow cytometric analysis of peripheral blood and draining lymph node cells suggested that both CD41 and CD81 T cell subsets had migrated into the nodes. This finding is consistent with previous adoptive cell transfer studies indicating that both CD41 and CD81 T lymphocyte subsets are mediators of DNFBinduced contact hypersensitivity (30, 31). By contrast, after stimulation with purified protein derivative of tuberculin, the large increase in T cell traffic through lymph nodes during the recruitment phase was mostly due to CD41 memory phenotype T lymphocytes (8). During the induction of contact hypersensitivity, abundant MIP-1a and MIP-1b mRNA and protein expression in the draining lymph nodes was detected. The kinetics of the protein expression of MIP-1a and MIP-1b was coincident with the recruitment of CD41 and CD81 T lymphocyte subsets, suggesting a functional role for these chemokines in the regulation of T cell recruitment. Although both chemokines were produced as early as 4 h after repainting, there was a Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 5. Mast cells are the predominant source of MIP-1b in draining lymph nodes during DNFB sensitization. A and B, Adjacent sections of the subcapsular region (2503 magnification) of a lymph node from a mouse sacrificed 4 h after DNFB treatment, showing expression of MIP-1b mRNA (blue staining with background neutral red counterstaining; A), which colocalized with cells immunostained for MMCP-5, indicative of mast cells (red staining with hematoxylin counterstaining; B). The arrows indicate at least two of the mast cells, which can confidently be identified in both sections. C and F, Adjacent sections of the subcapsular region of a lymph node from an acetone-treated (control) mouse illustrating the absence of MIP-1b mRNA expression (F), despite the presence of MMCP-5-positive mast cells (red staining; C) in the adjacent section. D, Lymph node section from a mouse sacrificed 24 h after DNFB treatment, immunostained with an anti-MIP-1b Ab showing widespread staining (in red) of the lumenal surface od endothelial cells (arrows). E, An adjacent section to D, stained with control goat IgG (negative control). 5670 CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES difference in the kinetics of expression, with MIP-1b protein expression being maximal at 4 h following DNFB repainting and persisting at high levels beyond 96 h, which was earlier and more prolonged than the expression of MIP-1a. Chemokine expression in the lymph nodes was not examined at time points in between the first and second paintings with DNFB; however, given the rapid kinetics of mRNA expression, it is likely that some induction of chemokine gene expression occurs before the second painting. The prominent expression of the protein, but not mRNA, of MIP-1b by endothelial cells in the DNFB-sensitized lymph nodes suggests translocation of this chemokine protein. This may occur via the recently described transendothelial chemokine transport mechanism (43), following initial synthesis by mast cells and macrophages in the nodes. In addition, the protein may be immobilized by proteoglycans on the surface of the endothelial cells to facilitate interaction with circulating lymphocytes (25, 44). Table II. Effect of neutralizing anti-chemokine Abs administered before sensitzation on the subsequent elicitation of contact hypersensitivity a Sensitization (%) Acetone DNFB DNFB DNFB DNFB (0.5) (0.5) (0.5) (0.5) Challenge (%) Acetone DNFB (0.2) DNFB (0.2) DNFB (0.2) DNFB (0.2) DNFB (0.2) Treatment Before Sensitization Ear Thickness (mm), Mean 6 SD Reduction (%) Anti-MIP-1a and 1b Goat IgG Sodium chloride 60 6 0.2 80 6 0.3 380 6 1.2 305 6 0.3* 365 6 0.2 NS 370 6 0.3 NS 19.7 3.9 2.6 a Five days after pretreatment with a combination of anti-MIP-1a and anti-MIP-1b or irrelevant Abs and sensitization with DNFB, mice were challenged by application of 10 ml of 0.2% DNFB in acetone olive oil to one ear. Control mice were similarly painted with the vehicle alone. The degree of ear swelling was measured before challenge and 24 h postchallenge by an independent observer, using an engineer’s micrometers (Mitutoyo). Each ear lobe was measured twice and contact hypersensitivity determined as the amount of swelling of the DNFB-challenged ear compared with the thickness of the vehicle-treated ear expressed in micrometers. Mice that were challenged with the hapten without previous sensitization served as an additional negative control. Each experimental and control group consisted of four animals. Results are expressed as mean 6 SD. *p , 0.05 vs positive control; NS, not significant (Two-tailed Mann-Whitney U test of the row data). Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 6. Chemokine inhibition reduces recruitment of T lymphocytes in the lymph nodes. Four hours before sensitization with DNFB, experimental animals were pretreated by i.p. injection of 500 mg of antiMIP-1a and/or anti-MIP-1b neutralizing Abs. Control animals were injected with control goat IgG in doses comparable to those given the experimental animals. Pretreatment with anti-MIP-1a and/or anti MIP-1b Abs significantly abrogated the recruitment of T lymphocytes into the draining lymph nodes. Mice similarly treated with the maximum amount (1 mg) of control goat IgG showed no inhibition of T cell accumulation after DNFB sensitization. Each experimental and control group consisted of four animals. Results are presented as mean 6 SD. The kinetics and magnitude of lymphocyte accumulation in the regional lymph nodes demonstrated following Ag challenge in this study were very similar to previously published reports (8, 9, 42). Although prior studies have indicated the involvement of draining lymph nodes in both the sensitization and elicitation phases of DNFB-induced contact hypersensitivity (30 –33), there are no reports on the histologic changes in the lymph nodes in this model. At the early time points, the nodes showed marked expansion of the subcapsular and medullary sinuses, which were filled with a large number of cells, predominantly lymphocytes, macrophages, and mast cells. This extensive expansion of the sinuses may reflect an increased flow of lymph and inflammatory cells from the skin via the afferent lymphatic vessels into the draining nodes. At later time points, increased activity of germinal centers was visible in some nodes, indicating a proliferative response to the Ag. The significance of MIP-1a and MIP-1b in this model was further confirmed by substantial inhibition of T lymphocyte recruitment into draining lymph nodes following administration of either anti-MIP-1a or anti-MIP-1b Abs. The reported preferential activity of these chemokines on CD41 or CD81 T lymphocyte subset was not observed, as the accumulation of both T cell subsets was similarly inhibited by either anti-MIP-1a or anti-MIP-1b Abs. However, the combination of both Abs produced a significantly greater reduction in CD81 ( p , 0.05) than CD41 T lymphocyte recruitment. This suggests that in CD41 T lymphocytes the two chemokines may act via a receptor (such as CCR5) that binds both ligands, whereas in CD81 T lymphocytes, additional receptors (such as CCR1, which is not responsive to MIP-1b) may also be utilized (27). Other recently identified b-chemokines such as secondary lymphoid tissue chemokine (SLC) and EBI1-ligand chemokine (ELC) have been proposed to be relevant to T lymphocyte recruitment into lymphoid organs (40, 45). These chemokines are constitutively expressed in lymphoid tissues and are chemotactic for lymphocytes (45). However, there are no in vivo data regarding the function of these molecules. 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Therefore, pretreatment of animals with a combination of anti-MIP-1a and anti-MIP-1b before hapten application not only significantly abrogates T cell recruitment to the draining nodes, but also partially blocks the subsequent delayed-type hypersensitivity reaction. This finding suggests that mice injected with anti-MIP-1a and anti-MIP-1b Abs followed by hapten application became partially tolerant to this hapten, perhaps as a result of decreased recruitment of haptenprimed T cells into the regional lymph nodes and thus a decrease in the expansion of hapten-specific T cells responsible for mounting a strong response upon subsequent challenge. A striking finding in this work is the demonstration of mast cells as the predominant cellular source of MIP-1b in the lymph nodes of DNFB-treated mice. These cells were distinctively located in the subcapsular and hilar regions of the nodes. Previous in vitro experiments have described the expression of MIP-1b mRNA by mast cell lines (46, 47). However, the abundant expression of this chemokine by mast cells observed in this in vivo model of contact hypersensitivity is novel. The brisk appearance of mast cells in a distinctive location in the subcapsular and hilar regions of the nodes, in association with the decrease in their number in the affected skin shortly after repainting with DNFB, strongly suggests that mast cells travel from the skin via the afferent lymphatic system. As expected, mast cell accumulation in the draining nodes was unaffected by neutralization of MIP-1a and MIP-1b activity,4 indicating that movement of these cells into the nodes is independent of a concentration gradient of these chemokines. The novel observation of mast cells as the dominant source of MIP-1b indicated in this work clarifies the controversy regarding the role of these cells in contact hypersensitivity (48 –51). In particular, mast cells are not only a source of histamine, serotonin, and other vasoactive amines that are believed to control vascular tone and permeability (52, 53), but also act as a key early regulator of T cell recruitment into draining lymph nodes. These findings provide the first direct in vivo evidence of chemokine regulation of trafficking of T lymphocyte subpopulations into lymph nodes during the induction of an immune response. Mast cells accumulating in the nodes have been identified as the principal source of MIP-1b in this model of contact hypersensitivity. Further studies examining additional chemokines and other models of immune response are warranted, as interventions to manipulate this chemokine-dependent T lymphocyte trafficking pathway may have profound influences on the outcome of host responses to infection or autoimmune triggers. 5671 5672 38. 39. 40. 41. 42. 43. 44. 46. prevents induction of contact hypersensitivity and induces hapten-specific tolerance. J. 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